Isolation And Molecular Biological Characterization Of Dendrolimus Punctatus Tetravirus

In the virus resource investigation of Dendrolimus, we have found a spherical icosahedral virus which is different from anyone of the reported viruses isolated from Dendrolimus. By the analysis of the physiochemical characteristics, serological relationship and genomic nucleotide sequences, we have drawn a conclusion that the virus is a new one that should be classified within the Omegatetravirus genus of the Tetraviridae and we have tentatively named it Dendrolimus punctatus tetravirus (DpTV).Through homogenization, difference-centrifugation, sucrose density gradient centrifugation, CsCl equilibrium-density gradient centrifugation, we have got the virions with high concentration. The buoyant density of DpTV particles in CsCl is 1.281 g/ml. When negatively stained with PTA, they appear icosahedral and nonenveloped, have a mean diameter of about 40 nm. Examination of DpTV capsid proteins by SDS-PAGE reveals that they are similar, but not identical, to those of NωV(Nudaurelia ω virus). DpTV has two capsid proteins: a major protein of 62,500 Da and a minor protein of about 6,800 Da. Nucleic acids extracted from DpTV particles are readily hydrolyzed by RNase A, but not by DNase I, under similar conditions, indicating they are single-stranded RNAs. The RNA preparation migrates as two species (RNA1 and RNA2) in agarose gel under denaturing conditions; the sizes of these are estimated as approximately 5,500 and 2,500 nucleotides (nt), respectively. These characteristics for DpTV most closely match those of the Omegatetravirus genus of the Tetraviridae. Immunoassay indicates that DpTV is serologically related to NωV and these two viruses are more closely related. This has further proved that DpTV is a new member of the Omegatetravirus genus of the Tetraviridae.In order to further investigate the genomic organization and classification of DpTV at the molecular level, we have determined the nucleotide sequences of DpTV RNA1 and RNA2 by using methods of RT-PCR, 3', 5'-RACE and cDNA synthesis in the presence of random primers. These two nucleotide sequences have been submitted to the Genbank nucleotide sequence database and have been assigned the accession numbers AY 594352 (RNA1) and AY 594353 (RNA2).The complete nucleotidesequence of DpTV RNA1 is 5,492 nt in length and has a G+C content of 57.3%; RNA2 consists of 2,490 nt and is cytosine-rich with 32.4% C, 23.5% G, 21.0% A, 23.1% T. The 3' ends of the genomic RNAs of DpTV are not polyadenylated and contain a terminal CCA box. Secondary structure analysis shows that both 3' terminal nucleotides of the DpTV RNAs can be folded into tRNA-like secondary structures which differ from the known plant virus tRNA-like structures in that it forms a pseudoknot in the aminoacylstem. Sequence analysis has identified a large open reading frame (ORF) on DpTV RNA1 encoding 1,649 amino acids (aa), starting with the first AUG (nt 37-39) and terminating at UGA (nt 4984-4986). This protein, named pi 80, had a calculated molecular weight of 179,474 Da and a calculated pi of 8.10. The nucleotide sequence of DpTV RNA1 is 61% identical to that of HaSV(Helicoverpa armigera stunt virus), although the HaSV RNA1 sequence is 180 bases shorter. Notably, the 3' non-coding sequence of DpTV RNA1 is the same length as that of HaSV (36 nt). DpTV RNA2 contains two ORFs (ORFpl7 and ORFp70) that are likely to be expressed during the replication of DpTV. These two ORFs, which partially overlap, begin at the first two AUG condons and locates between an untranslated leader sequences of 278 bases and a 3' non-coding sequence of 185 bases. ORFpl7 begins with the first AUG and encodes a predicted 158-aa, 16,801-Da protein with a calculated pi of 9.49. ORFp70 begins with the second AUG and encodes a protein of 643 aa, with a molecular weight of 69,744 Da and a calculated pi of 7.16. The nucleotide sequence of DpTV RNA2 is 74% and 63% identical to those of NcoV and HaSV, respectively, and is longer than both (42 and 12 nt, respectively).Analysis of pi7 using BLAST reveals that the amino acid sequence has no putative conserved domains, but has a high degree of overall identify (44%) to the pi 7 of HaSV Like the pi 7 of HaSV, the pi 7 of DpTV also has a high content (48.1%) of PEST amino acids, with 15.8% Pro, 3.8% Glu, 15.8% Ser, and 12.7% Thr and the function of this protein remains unknown.The p70 is the capsid precursor of DpTV. Multiple sequence alignment of the DpTV capsid precursor sequence with those of five other tetraviruses reveals that DpTV is more closely related to NcoV than to HaSV, PrV(Providence virus), N$V(Nudaurelia capensis P virus) and T&V(Thosea asigna virus). The DpTV capsid precursor shares 86% identity with that of NcoV, 66% with HaSV, 30% with PrV, 23% with NpV, and 21% with TaV. Although, Npv and NcoV infect the same host insects,NcoV is most closely related to DpTV, whereas NpV is almost the most distantly related of all the sequenced tetraviruses. The capsid protein precursor of DpTV might be cleaved once at an Asn-Phe site located after residue 570, producing two proteins with molecular masses of 62,126 Da and 7,634 Da. Like other tetraviruses, the capsid precursor of DpTV could be divided into four major domains: N-terminal domain, jelly-roll domain, Ig-like domain and C-terminal domain. The jelly-roll domain is consisted of 8 P-sheets and is divided into two segments by the central Ig-like domain. The C-terminal domain corresponds to the small peptide that might be released by cleavage.The pi80 is the replicase of DpTV. Like HaSV and NpV, the replication protein of DpTV contains the three major functional domains. These are, in order from the N terminus, the putative methyltransferase domain, the relicase domain and the polymerase domain. Alignment of the DpTV replicase with those of NpV and HaSV has revealed a few highly conserved residues within the motifs that define these domains. Homology analysis reveals that the evolutionary distance between the methyltransferase domains of DpTV and HaSV is less than that between DpTV and NPV, DpTV shows 67% identity to HaSV and 38% to NpV. The sequence of the RNA-dependent RNA polymerase domain of DpTV shared 76% identity with that of HaSV and 36% with NpV. Alignment of the amino acid sequences of the helicase domains shows that DpTV and HaSV are distantly related, with 63% identity. In contrast, NcoV is very closely related to HaSV, with 88% identity in this domain.The genomic organization and the homology of the capsid precursor and replication protein have also proved that DpTV is a new Nudaurelia ?-like viruse of the Tetraviridae at the molecular level.